Inhibitor of the differentiation of t cells into th1 cells

ABSTRACT

The present invention is to provide an inhibitor that inhibits differentiation of T cells into Th1 cells. 
     The inhibitor that inhibits differentiation of T cells into Th1 cells contains, as an active ingredient, pitavastatin or a salt thereof.

TECHNICAL FIELD

The present invention relates to a drug for inhibiting differentiationof T cells into Th1 cells, which plays a central role in the onset anddevelopment of, for example, multiple sclerosis, type I diabetes, orrheumatoid arthritis.

BACKGROUND ART

Lymphocytes include T cells, and B cells, which produce antibodies(immunoglobulins). T cells include helper T cells (CD4 antigenpositive), which regulate immune response to antigens presented bymonocytes or macrophages, and killer T cells (CD8 antigen positive),which destroy, for example, virus-infected cells. Helper T cells includeTh1 cells (T helper 1 cells) and Th2 cells (T helper 2 cells). WhetherTh1 cells (cellular immunity) or Th2 cells (humoral immunity) aredominant is determined by whether IL-12 or IL-4 is produced byantigen-presenting cells.

Thus, Th1 cells and Th2 cells respectively play an immunological role,and are involved in biological defense. However, it has been known toinduce differentiation of T cells into Th1 cells and then excessiveactivation of Th1 cells may cause various diseases. Diseases known to becaused by excessive induction of Th1 cells include multiple sclerosis,type I diabetes, and rheumatoid arthritis.

Among these diseases, multiple sclerosis is a cryptogenic, intractabledisease which is designated as a specified disease by the JapaneseMinistry of Health, Labor and Welfare, and this disease involves centraldysfunction due to cerebrospinal demyelination and causes sightproblems, dyskinesia, etc. In Europe and the United States, theprevalence of multiple sclerosis is higher than that of any otherneurological diseases affecting young adults, and is about 50 per100,000 population. In Japan, the prevalence of multiple sclerosis isestimated to be about 8 or 9 per 100,000 population, and the number ofmultiple sclerosis patients is estimated to be about 12,000. In manycases, multiple sclerosis involves repeated remissions and relapses(relapsing-remitting multiple sclerosis). Particularly, the onset ofmultiple sclerosis often occurs around age 30, and relapse of thedisease becomes less frequent with increasing age. However, somemultiple sclerosis patients exhibit rapid progression of symptoms(chronic progressive multiple sclerosis). It has been shown that T cellsor macrophages invade lesion sites of multiple sclerosis patients, andthese cells are considered to break myelin protein and myelin sheath incentral nerves, thereby causing neurological disorders (Non-PatentDocument 1). Demyelinating lesions may be distributed throughout thecentral nervous system, but are located mainly in, for example, theoptic nerves, the brain stem, the spinal cord, and the cerebellum.Multiple sclerosis causes a variety of symptoms (e.g., paralysis of thelimbs, shivering, fatigue, optic nerve disorder, dysuria, anddyschezia), and these symptoms vary depending on the site where nervesare damaged.

In current therapy for multiple sclerosis, a steroid is used in theacute phase, whereas an interferon-β (IFN-β) pharmaceutical product or aglatiramer pharmaceutical product is used for prevention of relapse.However, the latter pharmaceutical product is not approved in Japan. AnIFN-β pharmaceutical product acts on T cells, to thereby control theamount of interferon-γ produced, and to suppress antigen presentation byantigen-presenting cells (Non-Patent Document 2). Therefore,conceivably, an IFN-β pharmaceutical product suppresses enhancement ofimmune response, and contributes to delay of progression of multiplesclerosis symptoms or reduction in relapse frequency. However, even whensuch a pharmaceutical product is administered to multiple sclerosispatients, relapses of multiple sclerosis occur (average relapsefrequency: about 0.8 a year), and a method for reliably preventingrelapse of multiple sclerosis has not yet been developed (Non-PatentDocument 3). In addition, such a pharmaceutical product requiresfrequent subcutaneous administration, which causes problematic sideeffects such as fever and subcutaneous ulcer, and imposes a considerableburden on patients in terms of compliance. Therefore, development of apharmaceutical product which can be orally administered is consideredvery important for the treatment of multiple sclerosis. Meanwhile,although pulse therapy using cyclophosphamide (i.e., animmunosuppressive agent) has been reported to be effective for patientswith severe progressive multiple sclerosis, this agent causes severeside effects (e.g., leukopenia and alopecia), and thus requires carefuladministration. Under such circumstances, keen demand has arisen for aneffective therapeutic method for multiple sclerosis.

Statin compounds (i.e., HMG-Co reductase inhibitors) are known toexhibit various pharmacological effects. It has been known that, forexample, atorvastatin and simvastatin are effective for an experimentalautoimmune encephalomyelitis (EAE) model; exhibit the effect ofinhibiting proliferation of T cells and the effect of inducingdifferentiation of T cells into Th2 cells; and thus are probablyeffective for multiple sclerosis (Non-Patent Documents 4 and 5).

-   Non-Patent Document 1: Noseworthy, et al.; N. Engl. J. Med. Vol.    343, No. 13, pp. 938-52 (2000)-   Non-Patent Document 2: Joseph, et al.; J. Neuroimmunol. Vol. 20, No.    1, pp. 39-44 (1988)-   Non-Patent Document 3: The IFNB Multiple Sclerosis Study Group;    Neurology Vol. 43, No. 4, pp. 655-61 (1993)-   Non-Patent Document 4: Nature, 420, 78-84, 2002-   Non-Patent Document 5: Lancet, 363, 1607-1608, 2004

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

However, no studies have been conducted on the effects of pitavastatin(i.e., a statin compound) on multiple sclerosis or Th1 cells.

An object of the present invention is to provide a drug whichselectively inhibits induction of differentiation of T cells into Th1cells, and which is useful for the treatment of a disease caused byactivation of Th1 cells.

Means for Solving the Problems

In view of the foregoing, the present inventor has conducted extensivestudies focusing on pitavastatin (i.e., a statin compound), whoseeffects on multiple sclerosis or Th1 cells have not yet been elucidated,and as a result has found that pitavastatin or a salt thereof exhibitsthe effect of inhibiting differentiation of T cells into Th1 cells,which effect is about 10-fold higher than that of atorvastatin, and alsoexhibits the effect of inhibiting activation of Th1 cells. The presentinvention has been accomplished on the basis of this finding.

Accordingly, the present invention provides an inhibitor that inhibitsdifferentiation of T cells into Th1 cells, which contains, as an activeingredient, pitavastatin or a salt thereof.

The present invention also provides an inhibitor that inhibitsactivation of Th1 cells, which contains, as an active ingredient,pitavastatin or a salt thereof.

The present invention also provides a pharmaceutical composition forinhibiting differentiation of T cells into Th1 cells, the compositioncomprising pitavastatin or a salt thereof, and a pharmaceuticallyacceptable carrier.

The present invention also provides a pharmaceutical composition forinhibiting activation of Th1 cells, the composition comprisingpitavastatin or a salt thereof, and a pharmaceutically acceptablecarrier.

The present invention also provides use of pitavastatin or a saltthereof for producing an inhibitor that inhibits differentiation of Tcells into Th1 cells.

The present invention also provides use of pitavastatin or a saltthereof for producing an inhibitor that inhibits activation of Th1cells.

The present invention also provides a method for inhibitingdifferentiation of T cells into Th1 cells, the method comprisingadministering pitavastatin or a salt thereof to a subject in needthereof.

The present invention also provides a method for inhibiting activationof Th1 cells, the method comprising administering pitavastatin or a saltthereof to a subject in need thereof.

The present invention also provides pitavastatin or a salt thereof forinhibiting differentiation of T cells into Th1 cells.

The present invention also provides pitavastatin or a salt thereof forinhibiting activation of Th1 cells.

Effects of the Invention

Pitavastatin or a salt thereof exhibits an extremely-marked effect ofinhibiting differentiation of T cells into Th1 cells and anextremely-marked effect of inhibiting activation of Th1 cells.Therefore, pitavastatin or a salt thereof is useful as a therapeuticdrug for a disease caused by excessive induction of differentiation of Tcells into Th1 cells.

BRIEF DESCRIPTION OF THE DRAWINGS [FIG. 1]

FIG. 1 shows the effect of pitavastatin or atorvastatin on inhibitingactivation of Th1 cells (NK: pitavastatin, Atorva: atorvastatin).

[FIG. 2]

FIG. 2 shows comparison between pitavastatin and atorvastatin in termsof the effect of inhibiting differentiation of T cells into Th1 cellsand the effect of promoting differentiation of T cells into Th2 cells.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention employs pitavastatin(((3R,5S,6E)-7-[2-cyclopropyl-4-(4-fluorophenyl)-3-quinolyl]-3,5-dihydroxy-6-heptenoicacid): U.S. Pat. No. 5,856,336 and JP-A-1989-279866) or a salt thereof.As used herein, “pitavastatin or a salt thereof” also encompasses ahydrate of pitavastatin or a salt thereof, and a solvate of pitavastatinor a salt thereof with a pharmaceutically acceptable solvent. Examplesof the salt include alkali metal salts such as a sodium salt and apotassium salt; alkaline earth metal salts such as a calcium salt and amagnesium salt; organic amine salts such as a phenethylamine salt; andammonium salts. Of these, a salt of pitavastatin is preferred, and acalcium salt is particularly preferred.

Pitavastatin or a salt thereof may be produced through the methoddescribed in U.S. Pat. No. 5,856,336 or JP-A-1989-279866.

As described in the Examples hereinbelow, pitavastatin or a salt thereofexhibits the effect of considerably inhibiting induction ofdifferentiation of human T cells into Th1 cells. That is, pitavastatinor a salt thereof inhibits differentiation of T cells into Th1 cells,and markedly improves Th1/Th2 balance. Also, pitavastatin or a saltthereof strongly inhibits activation of Th1 cells. Such effects ofpitavastatin are 10-fold higher than those of atorvastatin, which, amongstatin compounds, has hitherto been suggested to be probably effectivefor multiple sclerosis. Thus, the effects of pitavastatin are so potentthat they cannot be predicted from those of atorvastatin. Therefore,pitavastatin or a salt thereof is useful as a therapeutic drug for adisease caused by differentiation of T cells into Th1 cells oractivation of Th1 cells, such as multiple sclerosis, type I diabetes, orrheumatoid arthritis.

No particular limitation is imposed on the dosage form of the drug ofthe present invention, and the dosage form may be appropriatelydetermined in consideration of the purpose of treatment. For example,the drug may be orally administered in the form of, for example, tablet,capsule, granule, film coating agent, powder, or syrup, or may beparenterally administered in the form of, for example, injection,suppository, inhalant, percutaneous absorption agent, eye drop, or nasaldrop. The drug is preferably provided in the form of a peroral product.

A pharmaceutical product suitable for such a dosage form may contain apharmaceutically acceptable carrier. Examples of the carrier includeexcipients and extenders such as starches, lactose, sucrose, mannitol,and silicic acid; disintegrating agents such as agar, calcium carbonate,potato or tapioca starch, alginic acid, and composite silicic acidsalts; binders such as hydroxypropylmethylcellulose, methylcellulose,sodium carboxymethylcellulose, alginic acid salts, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; lubricants such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodiumlaurylsulfate, and mixtures thereof; diluents such as lactose andcornstarch; buffers such as organic acids (e.g., citric acid, phosphoricacid, tartaric acid, and lactic acid), inorganic acids (e.g.,hydrochloric acid), alkali hydroxides (e.g., sodium hydroxide andpotassium hydroxide), and organic amines (e.g., triethanolamine,diethanolamine, and diisopropanolamine); antiseptics such asp-hydroxybenzoic acid esters and benzalkonium chloride; emulsifiers suchas anionic surfactants (e.g., calcium stearate, magnesium stearate, andsodium laurylsulfate), cationic surfactants (e.g., benzalkoniumchloride, benzethonium chloride, and cetylpyridinium chloride), andnonionic surfactants (e.g., glyceryl monostearate, sucrose fatty acidesters, polyoxyethylene hydrogenated castor oil, polyoxyethylenesorbitan fatty acid esters, polyoxyethylene fatty acid esters, andpolyoxyethylene alkyl ethers); and stabilizers such as sodium sulfite,sodium hydrogensulfite, dibutylhydroxytoluene, butylhydroxyanisole, andedetic acid. Optionally, the pharmaceutical product may appropriatelycontain an additional additive (e.g., a flavoring agent, a dispersant, apreservative, or a perfume) in combination with any of theaforementioned carriers.

In the present invention, the dose of pitavastatin or a salt thereof maybe appropriately determined in consideration of the body weight, age,sex, symptom, etc. of a patient in need thereof. Generally, the dailydose of pitavastatin or a salt thereof for an adult is 0.01 to 50 mg,preferably 0.1 to 20 mg, more preferably 1 to 10 mg. The daily dose isadministered once a day, or in a divided manner (several times a day).

Examples

The present invention will next be described in more detail by way ofexamples. However, the technical scope of the present invention is notlimited to these examples.

Example 1 (Method)

Five C57BL/6 mice were immunized with myelin oligodendrocyteglycoprotein (MOG), and mice with experimental autoimmuneencephalomyelitis (EAE) (i.e., a model of multiple sclerosis) wereprepared in a conventional manner. On day 14 after immunization, spleencells were collected from each mouse and cultured in a 24-well cultureplate coated with CD3 and CD28 antibodies, and then stimulated with MOGin the presence of pitavastatin or atorvastatin, to thereby activate Th1cells. The Th1 cytokine (IFN-γ) concentration of the supernatant of theresultant cell culture was determined through ELISA.

(Results)

As shown in FIG. 1, a concentration of 10 μM or more of pitavastatin(“NK” in FIG. 1) significantly reduced the amount of IFN-γ produced byTh1 cells derived from spleen cells of MOG-immunized mice; i.e.,inhibited activation of Th1 cells. As also shown in FIG. 1, 25 μM ofatorvastatin (“Atorva” in FIG. 1) significantly inhibited production ofINF-γ by Th1 cells.

Example 2 (Method)

Peripheral blood (10 mL) was collected from six healthy adults inheparin, and monocytes were isolated through the Ficol-Hypaque method(i.e., a customary method). The thus-isolated cells were diluted to1×10⁶/mL and then inoculated into a 24-well culture plate coated withCD3 and CD28 antibodies (Falcon), followed by culturing for four days inthe presence of IL-12 (100 ng/mL), to thereby induce differentiation ofT cells into Th1 cells. Atorvastatin or pitavastatin (0.01 to 10 μM) wasadded at the time of initiation of culturing, and the effect of eachdrug on differentiation of T cells into Th1 cells was examined. Aftercompletion of culturing, cells were washed thrice with PBS, andcytokines (IFN-γ and IL-4) in the cells and CD4 on the surfaces of thecells were stained in a conventional manner. The ratio of Th1(CD4-positive, IFN-γ-positive cells) or Th2 (CD4-positive, IL-4-positivecells) to all the CD4-positive T cells was determined through flowcytometry.

Specifically, lymphocytes were stimulated with anti-CD3 antibody andanti-CD28 antibody, and the amounts of INF-γ (Th1 cytokine) and IL-4(Th2 cytokine) in the cells were determined through flow cytometry, tothereby compare pitavastatin with atorvastatin in terms of the effect ofinhibiting differentiation of peripheral blood T cells into Th1 cells,and the effect of promoting differentiation of peripheral blood T cellsinto Th2 cells.

(Results)

As shown in FIG. 2, in the case of atorvastatin, at least 10 μM wasrequired to exhibit the effect of inhibiting differentiation of T cellsinto Th1 cells or the effect of promoting differentiation of T cellsinto Th2 cells. In contrast, 1 μM of pitavastatin significantlyexhibited the effect of inhibiting differentiation of T cells into Th1cells or the effect of promoting differentiation of T cells into Th2cells. These data indicated that the effects of pitavastatin ininhibiting differentiation of T cells into Th1 cells and in promotingdifferentiation of T cells into Th2 cells are 10-fold higher than thoseof atorvastatin.

The aforementioned data (the above-determined amount of INF-γ produced)indicated that the effect of pitavastatin on activation of Th1 cells is2.5-fold higher than that of atorvastatin on inhibiting activation ofTh1 cells (FIG. 1).

Comparison between pitavastatin and atorvastatin in terms of the effectof inhibiting differentiation of human T cells into Th1 cells and theeffect of promoting differentiation of human T cells into Th2 cellsshowed that pitavastatin inhibits differentiation of T cells into Th1cells or promotes differentiation of T cells into Th2 cells 10-fold morestrongly than does atorvastatin; i.e., the Th1/Th2 ratio improvingeffect of pitavastatin is 10-fold higher than that of atorvastatin (FIG.2).

1. An inhibitor that inhibits differentiation of T cells into Th1 cells,which contains, as an active ingredient, pitavastatin or a salt thereof.2. An inhibitor that inhibits activation of Th1 cells, which contains,as an active ingredient, pitavastatin or a salt thereof.
 3. Apharmaceutical composition for inhibiting differentiation of T cellsinto Th1 cells, the composition comprising pitavastatin or a saltthereof, and a pharmaceutically acceptable carrier.
 4. A pharmaceuticalcomposition for inhibiting activation of Th1 cells, the compositioncomprising pitavastatin or a salt thereof, and a pharmaceuticallyacceptable carrier.
 5. Use of pitavastatin or a salt thereof forproducing an inhibitor that inhibits differentiation of T cells into Th1cells.
 6. Use of pitavastatin or a salt thereof for producing aninhibitor that inhibits activation of Th1 cells.
 7. A method forinhibiting differentiation of T cells into Th1 cells, the methodcomprising administering pitavastatin or a salt thereof to a subject inneed thereof.
 8. A method for inhibiting activation of Th1 cells, themethod comprising administering pitavastatin or a salt thereof to asubject in need thereof.